PROJECT SUMMARY/ABSTRACT With proteomics entering the third decade, the identification and quantification of primary protein sequences has become a mature science. However, many analytical challenges remain during the mapping of post-translational modifications (PTMs) of proteins, especially in their native state. For example, complete deciphering of the histone code entails the identification and localization of the different PTMs using top down strategies. While some advances have been made, traditional LC-MS/MS methods cannot fully separate histones with isomeric PTM position variants; this leads to the need to further develop new, fast, and orthogonal separations that can be easily integrated with mass spectrometry for the characterization of the ?histone code?. In the present project, we aim to test the hypothesis that ?PTMs induce structural changes in histones, allowing their separation and identification by the difference in ion mobility properties (particularly, for positional isomers), and fragmentation patterns?. Understanding the effect of PTMs on histone structure and function is central to the epigenetic regulation. Thus, to enable further advances in epigenetics, we will develop new multidimensional ion mobility separations for top- down isoform histone analyses. The ultimate goal of this project is to bring together technology experts in post-ionization, orthogonal separations and top-down mass spectrometry to develop an integrative, multidimensional analytical platform, capable of characterizing the ?histone code? using native, as opposed to proteolytically digested, histones. To accomplish this goal, we will pursue the following aims: 1) To integrate multi-stage, non-linear, and linear ion mobility separations with top-down mass spectrometry (IMSn-MS/MS); 2) To develop liquid chromatography (offline and online) strategies compatible with IMSn-MS/MS.; and 3) To evaluate histone PTM abundances in biological systems using LC-MS/MS and LC-IMSn-MS/MS strategies. To support these aims, major technological breakthroughs in each of the integrated areas will be achieved: i) non-linear IMS (with 3 new FAIMS geometries implemented and evaluated), ii) linear IMS (with a higher resolution, larger mobility range, and higher sensitivity TIMS cell implemented and evaluated), and iii) non-ergodic top-down fragmentation (i.e., ExD and UVPD) compatible with online, native LC and mobility workflows. Completing the aims of this proposal will provide new innovative and enabling analytical solutions and instrumentation, that will benefit the proteomics community at large and open new doors for functional top-down proteomics.